Flow cell apparatus

Abstract: A flow cell apparatus for measuring particles suspended in a liquid comprises a sample liquid, supply device, a sheath liquid supply device, a flow cell which has a flow passage passing through a transparent measuring portion, and a nozzle for causing the sample liquid, from the supply device to flow in the flow passage of the flow cell. The nozzle has at least one discharge port which is disposed in the flow passage in a spaced relation from the inner wall of the flow cell flow passage. The sheath liquid supply device causes the sheath liquid to flow around the nozzle discharge port and surround the sample liquid flow to form a sheath flow. The flow passage of the flow cell and the nozzle are so formed as to ensure a sample liquid flow of a fixed width through the measuring portion. A stable sample flow having a fixed width can be formed in the measuring portion even when the flow is at a high velocity, and particles in the sample liquid can efficiently be photographed with high accuracy.

Description: The present invention relates to a flow cell apparatus for obtaining morphological information of particles suspended in a liquid to analyze the information. A typical example of observing shapes of particles contained in a liquid to classify and analyze the particles is a urine sedimentation examination. The urine sedimentum examination is a morphological examination of particles in urine. Hitherto, employed for the urine sedimentation examination is by a visual method of dyeing sediment to make a specimen on a slide glass plate and inspecting the specimen with a microscope. The sediment contains particles of various sizes from several micrometers, such as blood corpuscle cells and bacilli, to hundreds of micrometers, such as columnar particles. These particles are observed by switching the magnification of the microscope between a high magnification and a low magnification. According to the foregoing method, typically, condensed urine in a quantity corresponding to natural urine of 5 microliters in a case of observation at a high magnification and or in a quantity corresponding to natural urine of 750 microliters in a case of a low magnification is observed to count the number of each sediment component, though the quantity of the sample to be observed varies depending upon the purpose. These has been another method which utilizes a flow cell having a flow passage of a specific form with a measuring portion provided therein to classify and analyze particles in a liquid. This method is what forms, in the flow passage of a flow cell, a flow of the liquid to be measured, which is surrounded by a sheath flow, and measures particles in the sample liquid by an optical means. Such a method is suitable to automate the examination of particles in a liquid. For example, National Publication of Unexamined Application No. 57-500995 has disclosed an apparatus in which a fluid sample is introduced into a flow passage of a specific shape and particles in the sample are photographed in a wide imaging region. The apparatus has a CCD camera connected to a microscope and a pulse light source for periodically emitting light in synchronization with the operation of the CCD camera, so that enlarged still images of particles even flowing can be taken with the CCD camera. Through analysis of the still images thus obtained, the particles in a liquid can morphologically be analyzed. Further, the concentration of the particles may be analyzed by counting how many the particles are contained in a volume of the photographed sample. Another method using a flow cell has been suggested, in which condition of a sample flow are varied during measurement of the same kind sample to change the state of the sample flow to measure the same. According to this method, the sample can be measured under a plurality of different conditions. An apparatus has been disclosed, as an example of such measurement, in Japanese Patent Unexamined Publication No. 3-105235. The apparatus is what utilizes the flow cell capable of forming a flat sample flow to measure particle components in urine. The above flow cell is so formed that a flow passage in a measuring portion is constant in size in the direction of thickness of a sample flow while gradually widening in the widthwise direction toward the downstream. The sample flow is widened in the widthwise direction thereof when it passes through the enlarged flow passage, to be decelerated and form a flat flow. The flow thus formed is thin in the direction of thickness of the flow while being wide in the widthwise direction thereof, and particles contained in a part of the flat sample flow are photographed. In the measurement described in Japanese Patent Unexamined Publication No. 3-105235, a microscope is switched between a high magnification mode and a low magnification mode to measure particles of various sizes. At this time, by changing the flow rate of the sample liquid or the sheath liquid to be supplied to the flow cell, the thickness of the sample flow can be controlled. That is, since the depth of focus of the microscope decreases when the sample flow is measured at a high magnification, the thickness of the sample flow is reduced so that the microscope can be focused on particles. In the case of a low magnification, the control is made contrarily. When using such an apparatus as disclosed in National Publication No. 57-500995 in an examination for a liquid having a low concentration of particles, such as an examination of urine sedimentation, the sample must be subjected to centrifugation. This is because that the concentration of the particles is too low and, unless centrifugation of the sample, mout of the images taken will be wasteful with no images of particles photographed therein. For instance, particles contained in urine are usually one or less per microliter and, without condensation of urine, there will be a case where no particle is photographed in 100 visual fields even at a low magnification. Accordingly, in order to accurately analyze a sample of a low concentration that contains only few particles to be analyzed, the volume of the sample to be analyzed must be increased to increase the number of particles to be measured. To efficiently perform such measurement, therefore, it is desirable to increase the velocity of the sample flowing in the flow cell. However, in the enlarged flow passage disclosed in National Publication No. 57-500995 which becomes wider in the measuring portion when the velocity is increased, there is a fear that the sample flow will fall into turbulence not to be uniform in thickness and to be uneven in the velocity distribution. This is disadvantageous when photographing particles with accuracy in the visual field of the measuring portion. Further, the sample flowing through the flow passage becomes a flat flow having an elliptic cross sectional shape, and the volume of the sample in the visual field of the measuring can hardly be stable. When a part of the sample flow is photographed for measurement, there will be a case where some particles pass out of the visual field for the measurement and accurate information of the particles and that of the concentration of the sample cannot be obtained. As described above, according to the conventional method as disclosed in National Publication No. 57-500995, the volume of a sample that can be analyzed within a given period of time is very small, and therefore, it is difficult to accurately obtain many images of particles in a low concentration sample within a given period of time. Moreover, in order to measure a sample under various conditions, it is desirable to arbitrarily control the shape of a sample flow. In the apparatus disclosed in Japanese Patent Unexamined Publication No. 3-105235, a sample liquid of a circular cross section discharged through a columnar nozzle is once squeezed and then laterally widens to be flat while decreasing its velocity in the enlarged flow passage. In this case, since only the flow rate of the sample liquid and that of the sheath liquid are parameters for controlling the state of the sample flow, it is difficult to individually and arbitrarily control the flow velocity of a central portion of the sample flow and the thickness of the sample flow. On the other hand, in the above apparatus, when the flow rate of the sample liquid and that of the sheath liquid are set to cause the flow velocity of the central portion of the sample flow and the thickness thereof to be desired values, the width of the sample flow and the flow velocity of peripheral portions thereof cannot be controlled. That is, the width of the sample flow and the flow velocity of the peripheral portions of the sample flow depend on the shape of the fluid passage, and they cannot be controlled. In the apparatus disclosed in Japanese Patent Unexamined Publication No. 3-105235, the sample flow is laterally widened in the enlarged, decelerating flow passage to be a flow which is wider than a range required to measure the sample flow, and a part of the sample flow is photographed. As a result, there will be a case where the particle image becomes broken at an end of the photographed image. Further, the flow velocity becomes uneven between the central portion and the peripheral portion of the sample flow. As described above, according to the conventional method disclosed in Japanese Patent Unexamined Publication No. 3-105235, it is difficult to individually, actively and arbitrarily control the shape and the flow velocity of the sample flow in the flow cell. US-A-5 007 732 discloses a flow cell device comprising a sheath fluid inlet, a sample fluid inlet, and a flow passage which communicates with the sheath fluid inlet and contracts downward. Furthermore the flow cell device according to US-A-5 007 732 comprises a straight capillary flow passage communicating with said flow passage. A discharge port is provided at a terminal end of the capillary flow passage, and a nozzle is opened in the flow passage in the same direction as the direction of flow of the sample fluid in the capillary flow passage. The capillary flow passage and the flow passage have substantially rectangular cross-sections. A top wall and a bottom wall of the capillary flow passage are made transparent so that measuring light can pass therethrough. One side wall of the capillary flow passage has a smooth surface, while the other side wall of the capillary flow passage has a roughened surface. The flow of the fluid in the capillary flow passage is a laminar flow having a velocity gradient. A further flow cell apparatus for measuring particles suspended in a liquid is known from US-A-4 983 038. The flow cell apparatus according to US-A-4 983 038 comprises means for supplying a sample liquid to be measured, means for supplying a sheath liquid, flow cell means for defining a flow passage with a transparent measuring portion providing an unobstructed view of the flow passage in at least one transversal direction thereof, nozzle means provided in fluid communication with the sample liquid supplying means and comprising a nozzle body, and at least one discharge port formed at one end of said nozzle body and disposed in the flow passage in a spaced relation from an inner wall of the flow passage for causing the sample liquid to flow into the flow passage of said flow cell means, and guide plates disposed on first opposite sides of said discharge port for guiding the sample liquid from said discharge port, whereby the sheath liquid supplying means are so arranged in fluid communication with the flow passage of said flow cell means at a position upstream said discharge port with respect to the flow of the sample liquid as to cause the sheath liquid to surround the sample liquid from the discharge port to form a sheath flow, said nozzle body end is tapered to said discharge port to have on second opposite sides thereof tapered surfaces, whereby said second opposite sides are different from said first opposite sides, and said guide plates are tapered to their ends in downstream direction with respect to the flow of the sample liquid. The tapered surfaces of the nozzle body terminate at one end of said guide plates, whereby the corresponding arrangement is such that the tapered surfaces of the nozzle body slant in line with the guide plates. The flow passage of the flow cell apparatus according to US-A-4 983 038 greatly decreases in width. Therefore the sample liquid flow is greatly constricted after it leaves the nozzle, and the width of the sample liquid flow is reduced. Since the flow cell apparatus according to US-A-4 983 038 is designed for analyzing particles by means of a laser beam, the sample liquid flow has to be constricted to a narrower width than that of the laser beam. EP-A-0 556 971, which is prior art according to Article 54(3) EPC, discloses a flow cell apparatus for measuring particles suspended in a liquid, comprising means for supplying a sample liquid to be measured, means for supplying a sheath liquid, flow cell means for defining a flow passage with a transparent measuring portion providing an unobstructed view of the flow passage in at least one transversal direction thereof, nozzle means provided in fluid communication with the sample liquid supplying means and comprising a nozzle body, and at least one discharge port formed at one end of said nozzle body and disposed in the flow passage in a spaced relation from an inner wall of the flow passage for causing the sample liquid to flow into the flow passage of said flow cell means, and guide plates disposed on first opposite sides of said discharge port for guiding the sample liquid from said discharge port, whereby the sheath liquid supplying means are so arranged in fluid communication with the flow passage of said flow cell means at a position upstream said discharge port with respect to the flow of the sample liquid as to cause the sheath liquid to surround the sample liquid from the discharge port to form a sheath flow, and said guide plates are tapered to their ends in downstream direction with respect to the flow of the sample liquid. The nozzle body terminates at said guide plates, whereby the guide plates at the corresponding contacting faces have the same height as the height of the discharge port. It is an object of the invention to provide a flow cell apparatus capable of forming a stable, flat and high-speed sample flow which is less in turbulence so that one can accurately and efficiently obtain many particle images even when liquid to be measured has a low concentration of particles suspended therein. This object is achieved by a flow cell apparatus for measuring particles suspended in a liquid, comprising means for supplying a sample liquid to be measured, means for supplying a sheath liquid, flow cell means for defining a flow passage with a transparent measuring portion providing an unobstructed view of the flow passage in at least one transversal direction thereof, nozzle means provided in fluid communication with the sample liquid supplying means and comprising a nozzle body, and at least one discharge port formed at one end of said nozzle body and disposed in the flow passage in a spaced relation from an inner wall of the flow passage for causing the sample liquid to flow into the flow passage of said flow cell means, and first guide plates disposed on first opposite sides of said discharge port for guiding the sample liquid from said discharge port, whereby the sheath liquid supplying means are so arranged in fluid communication with the flow passage of said flow cell means at a position upstream said discharge port with respect to the flow of the sample liquid as to cause the sheath liquid to surround the sample liquid from the discharge port to form a sheath flow, said nozzle body end is tapered to said discharge port to have on second opposite sides thereof tapered surfaces, whereby said second opposite sides are different from said first opposite sides, said first guide plates are tapered to their ends in downstream direction with respect to the flow of the sample liquid, the tapered surfaces of the nozzle body terminate between said first guide plates, whereby the first guide plates are higher than a height of the discharge port at the terminating end of the nozzle body to stabilize a sample liquid flow from the discharge port at a constant width in at least a direction perpendicular to a direction of the unobstructed view in said measuring portion, and the flow passage has a constant width for preventing change in the width of the sample liquid flow. The flow cell apparatus of the invention is able to form a sample flow of a constant width in the measuring portion, which sample flow is stable even at a high speed. Thus, it is possible to carry out efficient measurements at the sample liquid with high accuracy. The sample flow in the flow cell apparatus according to the invention flows stably. Changes and irregularities in thickness and width of the flow are prevented. A sample flow having a uniform flow rate distribution in the widthwise direction is realized. In addition, since the sample liquid can continuously be supplied to the flow cell apparatus in a short time, many kinds of samples can successively be measured. Preferably said flow cell means further includes means provided upstream said measuring portion for narrowing the sheath flow in the unobstructed view direction of said measuring portion to increase a velocity of the sample liquid flow in the sheath flow and reduce a thickness of the sample liquid in the unobstructed view direction to form a flat flow. In this way the flow can be further stabilized and a flat sample flow of a cross-section having a large aspect ratio can be formed. Therefore, when particles in the sample liquid are photographed by a CCD camera connected to a microscope, pictures of them can be taken within the range of a photographing visual field while being correctly focused on. As a result, the accuracy in photographing can be improved. In a preferred embodiment of the flow cell apparatus according to the invention said nozzle means further includes means for changing the width of the sample liquid flow in accordance with a mesuring range in said measuring portion. Thus, the sample flow in the flow cell can be said to have an optimum width for the imaging portion since the width can be switched when the photographing magnification is changed. Moreover, sample flows having widths, thicknesses and flow rates suitable to plural types of examination conditions can successively be formed in the same flow cell. Therefore, examination can be performed precisely, the liquid passage system and the examination optical system can be simplified, the quantity of the sample required to perform an examination can be reduced, and the time required to complete the examination can be shortened. In addition, samples of plural types having widths, thicknesses and flow rates that suit the characteristics of the samples can succesively be formed in the same flow cell. Therefore, an examination can be performed under the most suitable conditions for the examination optical system. Accordingly, the examination can be performed precisely. Further preferred embodiments of the flow cell apparatus according to the invention are subject matter of claims 4 to 19. Embodiments of the flow cell apparatus according to the invention will now be described as examples with reference to the accompanying drawings in which: Fig. 1 is a perspective view showing the overall structure of a flow cell apparatus according to a first embodiment of the invention; Fig. 2 is an enlarged view for explanation of the connection between a flow cell and a sample fluid supply portion in the apparatus shown in Fig. 1; Fig. 3 is an exploded perspective view showing an essential structure of the flow cell shown in Fig. 1; Fig. 4 is an exploded perspective view of a casing in the flow cell of Fig. 3; Fig. 5 is an exploded perspective view of a nozzle in the flow cell of Fig. 3; Fig. 6 is a perspective view showing a flow in the flow cell of Fig. 3, whereby the nozzle-guide-arrangement in Fig. 6 is not in accordance with the present invention; Fig. 7 is a perspective view showing a flow in a flow cell according to a modification, whereby the nozzle-guide-arrangement in Fig. 7 is not in accordance with the present invention; Fig. 8 is an enlarged plan view showing a measuring portion in the flow cell of Fig. 6; Figs. 9A to 9D are enlarged plan views illustrating methods of changing an imaging visual field in the flow cell and the way of causing the sample fluid to flow, respectively; Fig. 10 is a schematic perspective view of a flow cell according to a modification for putting the method of Fig. 9D into Dractice; Fig. 11 L is an exploded perspective view showing the structure of a nozzle in the flow cell of Fig. 10; Fig. 12 is an exploded perspective view showing the structure of a nozzle according to another modification; and Figs. 13 and 14 are perspective views showing flows of different shapes by the nozzle of Fig. 12, respectively. The basic structure of a flow cell apparatus according to the invention will now be described with reference to an embodiment shown in Figs. 1 to 9. The flow cell apparatus shown in these figures exhibits the basic characteristic features of the invention and some additional features. The flow cell apparatus comprises a flow cell assembly and a means for supplying a sample. As shown in Fig. 1, the means for supplying a sample to a flow cell, which will be described later, is mainly composed of a pipette drive unit 29, a pipette arm 28 which is moved vertically and rotated by the pipette drive unit 29, and a pipe-shaped pipette 27 which is provided at a free end of the arm 28 to extract and supply a sample 26. As shown in Fig. 2, an O-ring 32 is attached to an end of the pipette 27 to improve the air tightness with a connection portion 5 of the flow cell. A sample suction port 33 in an end of the pipette 27 is tapered in order to prevent falling of the sample. Accordingly, the end of the pipette 27 and the connection portion 5 can be joined in intimate contact to each other to prevent leakage of the sample 26. In this embodiment, the sample supply means is constructed as described above. Therefore, a sample can continuously and quickly be supplied to the flow cell serving as a measuring section, and the embodiment has an advantage of being able to adapt to a case where many kinds of samples are successively measured. Further, the sample is not required to be introduced into the measuring section through a tube or the like, and accordingly it can directly be supplied from a position adjacent to the measuring section. Therefore, time taken to form a sheath flow in the flow cell can significantly be shortened. In the case where a sample is transported for a relatively long distance to the measuring section through a tube or the like, the overall passage, through which the sample has passed, must be cleaned. However, this embodiment, because of having the very short sample passage as described above, enables the area, that must be cleaned, to considerably be reduced. Further, cleaning can be performed by directly inserting the end of the pipette 27 into a cleaning tank 31, so that time required to complete the cleaning can be shortened. The flow cell assembly according to this example is constituted as follows. As shown in Fig. 3, a holder 4 has a seat formed on the bottom thereof. One of flanges 15 for holding a casing 2 which defines a flow passage is joined into intimate contact to the seat while being sealed with an O-ring 9. The other flange 15 is joined into intimate contact to a sheath liquid supply portion 11 while being sealed with an O-ring 9, and the sheath liquid supply portion 11 is installed to the holder 4. The sheath liquid supply portion 11 is provided, in the outer periphery thereof, with sheath liquid ports 7 for supplying a sheath liquid, and these ports are connected to a passage formed through the center of the supply portion 11. The central passage of the supply portion 11 communicates with the flow passage of the casing 2. A sample supply portion 10 is attached in intimate contact to the sheath liquid supply portion 11 while being sealed by another O-ring 9. The sample supply portion 10 is provided with a nozzle 3 serving as an outlet of the sample and the connection portion 5 through which the sample is supplied into a fluid passage for the nozzle 3. The connection portion 5 has a cleaning fluid suction port 6 provided for sucking waste fluid that has cleaned the flow cell. Also in the holder 4, a waste fluid port 8 for discharging waste fluid is formed. The nozzle 3 of the supply portion 10 is inserted into the flow passage of the casing 2 through the central passage of the supply portion 11. The casing 2, as shown in Fig. 4, comprises front plates 13, side plates 14 and flow contraction plates 12, all of which are made of a transparent material so that a flow of the sample in the flow passage is optically measured from outside. The flow passage in the casing 2 is defined by the elongated and flat front plates 13 and the side plates 14 with respective two plates of the same kind combined to each face the associated plate in such a manner that the width and the thickness of the flow passage are made constant. The flow contraction plate 12, which are in a trapezoidal shape thickened substantially at its lengthwise middle portion, are attached in the flow passage to face each other, so that the flow passage is partially narrowed in the direction of the thickness thereof. The front plates 13, the side plates 14 and the flow contraction plates 12 are made of, for example, glass, and assembled by optical bonding without deteriorating their transparency. The front plates 13, the side plates 14 and the contraction flow plates 12 are further secured by means of the flanges 15 at the upper and lower ends of the flow passage. As shown in Fig. 5, the nozzle 3 comprises a nozzle body 17 with two guides 16 joined respectively on the both sides of the nozzle body, which are longer than the nozzle body and each have a tapered end. The nozzle body 17 is directly fixed to the sample supply portion 10, a sample discharge port 19 of a substantially square cross in the nozzle body is in communication with the connection portion 5 through the sample passage 18, and the sample liquid injected through the connection portion 5 is discharged from the sample discharge port 19. The nozzle body 17 is machined and manufactured from a stainless material by, for example, a wire discharge process. The guide 16 are joined with high accuracy by, for example, a plating joining process. With the flow cell assembly thus formed, in which the casing 2 is attached to the holder 4 and the sample supply portion 10 with the nozzle 3 is attached to the sheath liquid supply portion 11 which is fixed to the holder 4, the embodiment has an advantage that adjustment, such as position aligning of the nozzle 3 and the casing 2, can be easily performed. Further, disassembling and cleaning of the casing 2 and the sheath liquid supply portion 11, that are considered to be contaminated most frequently, are facilitated, making the maintenance easy. Supply of a sample to the flow cell 1 (see Fig. 6) is carried out as follows by the foregoing sample supply means. First, the pipette arm 28 is moved vertically and rotated by the pipette drive unit 29 so that the pipette 27 is inserted into a sample container 30 in which the sample 26 is stored. Then, a syringe (not shown in the figures) connected to the pipette 27 is operated to suck the sample 26 of a predetermined quantity into the pipette 27. After that, the pipette 27 is lifted and-rotated to be moved to the position above the connection portion 5. The end of the pipette 27 and the connection portion 5 are brought into intimate contact to be connected to each other, then, the syringe is operated to discharge the sample 26 of a certain quantity from the connection portion 5 into the flow cell 1 at a regular rate, and the sample 26 is measured in the flow cell 1. At this time, as the connection portion 5 is directly connected to the sample passage 18, the sample 26 discharged from the pipette 27 passes through the sample passage 18 to be supplied into the flow cell 1 from the nozzle 3. Simultaneously, the sheath liquid is supplied to the ports 7 of the sheath liquid supply portion 11 and flows into the flow cell 1 through the central passage of the supply portion 11 to surround the nozzle 3. The flow passage in the flow cell 1 is formed as shown in Fig. 6 to have a parallel flow passage portion 1a, a flow contraction passage portion 1b, a flow passage portion 1c for measurement including an imaging portion 21, and a flow rate decelerating passage portion 1d. However, it is clearly stated that the nozzle-guide-arrangement shown in Fig. 6 is not in accordance with the present invention. The sample liquid containing particles 20 to be examined is supplied in a flowing direction 25 and discharged into the flow passage from the nozzle 3 in a predetermined quantity. On the other hand, the sheath liquid which is a clean liquid containing no particles is supplied in a flowing direction 24 into the flow passage around the nozzle 3. A sheath flow, that is a steady laminar flow in which the sheath liquid surrounds the sample liquid, is formed in the flow cell 1, and a sample flow 23 passes through the parallel flow passage portion la of the flow cell 1 at a constant speed. The sample flow 23 in the sheath flow flows while having a thin, wide and flat cross section due to pressure applied from the upper and lower sheath flows. The flow cell passage of the embodiment is formed in a shape, which is monotonously decreased in cross sectional area only in the direction of the thickness thereof from the sample discharge port 19 to the imaging portion 21 due to the provision of the flow contraction plates 12 described above, and it has the flow contraction passage portion 1b. In this flow contraction passage portion, the sheath flow monotonously increases its flowing speed. With this flow speed increasing passage portion, it is possible to form a stable flow, and therefore, a sample flow at a high speed more than 1000 mm/second can be formed. Further, since the sample flow 23 is able to stably flow and wakes in the directions of the thickness and the width are reduced, an improvement in the imaging accuracy can be realized. Moreover, the sample flow discharged from the nozzle 3 is uniformly contracted or reduced in size in the direction of the thickness thereof to form a thinner and uniform flat flow, and therefore, a further improvement in the imaging accuracy can be realized. The measurement passage portion lc follows the foregoing passage portion 1b, in which the thickness of the fluid passage is made substantially constant. The sample flow 23 is formed into a flat shape, the cross section of which has a very large aspect ratio such that the width of the sample flow 23 is about 200 to 300 micrometers and the thickness is about 5 to 20 micrometers. The flow passage in the imaging portion 21 is formed in a cross sectional shape which is sufficiently large in size in the widthwise direction as compared with its size in the direction of the thickness thereof, and the width of the flow passage is not changed from the outlet port of the nozzle to the measuring portion. Therefore, the sample flow 23 can be realized to be substantially constant in width and uniform in the flow rate distribution in the direction of the width thereof. The sheath flow then passes through the flow decelerating passage portion 1d and is discharged from the waste fluid port 8 of the holder 4. The particles 20 to be examined, which are contained in the sample flow 23, are photographed in an imaging visual field 21 by, for instance, a CCD camera connected to a microscope, so that they can be received as image information. The sample flow 23 is in the shape which is small in thickness and large in width. Such a flat shape is suitable easy to be focused on in the range of the imaging visual field 21 when the particles 20 to be examined are imaged by the CCD camera connected to the microscope, and therefore, it is suitable for photographing. For this reason, even in case of a microscope which is shallow in depth of focus, the particles 20 can be imaged in a condition of being correctly focused thereon. The nozzle 3 is provided with guides 16 which prevent turbulence of the flow of the sample liquid at the sample discharge port 19 and serve to keep the sample flow having a constant width. That is, the width of the sample flow 23 depends on the distance between the two guides 16 of the nozzle 3, and is kept substantially constant from the nozzle 3 to the imaging portion 21 because the width of the flow passage is made constant. Thus, through adjustment of the distance between the guides 16, the sample flow 23 can be set at a suitable width which is optimum for the imaging portion 21. After the measurement has been completed, the end of the pipette 27 is moved away from the connection portion 5 and is then inserted into a cleaning tank 31. In the cleaning tank 31, a cleaning liquid is radially applied to the end of the pipette 27 to wash out the sample on the pipette 27. Further, a cleaning liquid is supplied also from the inside of the pipette 27 and discharged, together with the sample removed from the interior, into the cleaning tank 31. Since the pressure in the sample liquid passage 18 is somewhat higher than that of the outside air when the end of the pipette 27 is separated from the connection portion 5, the sheath liquid is caused to rise through the sample liquid passage 18 to the connection portion 5 in a state where the pipette 27 is disconnected after the measurement has been completed. The connection portion 5 has the waste liquid suction port 6 (see Fig. 2) provided for sucking the sheath liquid that has cleaned the connection portion 5, and the sheath liquid is sucked through the waste liquid suction port 6, so that the sample adhered to the connection portion 5 is cleaned by the sheath liquid thus sucked. The flow cell 1 shown in Fig. 6 forms the parallel flow passage portion 1a. As shown in Fig. 7, however, another structure may be employed in which the parallel flow passage portion la is omitted from the flow cell 1, the other structural portions are the same as those shown in Fig. 6, and the nozzle 3 is disposed in the flow contraction passage portion 1b. However, it is clearly stated that the nozzle-guide-arrangement shown in Fig. 7 is not in accordance with the present invention. With such a structure, reduction or contraction of a flow is carried out in the flow contraction passage portion 1b at the same time when shaping of the flow is made between the guides 16, so that the sample flow is efficiently formed. Incidentally, in the embodiments and modifications described herein, similar component parts will be denoted by the same reference numerals, and description thereof will be omitted. The width of the sample flow 23 and the imaging region 21 will now be described in detail with reference to Fig. 8. The flow cell according to the embodiment shown in Fig. 6 is adapted to detect, by means of an optical system (not illustrated) whether or not the sample particles have passed through the upstream of the imaging portion 21 of the flow cell. That is, the structure is so constructed that a region indicated by reference numeral 22 is irradiated with, for example, laser beams, and dispersed light from the sample particles is detected. The particles contained in the sample liquid are irradiated with the laser beams when they pass through a particle detection region 22a or 22b so that passing of the particles is detected. The particles thus detected are photographed and recorded by an imaging region 21a or 21b. The flow cell according to this embodiment is so formed that the magnification of photographing the sample particles can be selected from a plurality of magnifications. Therefore, particles contained in the sample and having various sizes, such as large particles of about 100 microns to small particles of several microns, can accurately be photographed. For example, in a mode of photographing particles at a large magnification, they are detected in the particle detection region 22a, and their images are photographed in the enlarged visual field 21a. In another mode in which the particles are photographed at a relatively reduced magnification, they are detected in the particle detection region 22b, and their images of the particles are photographed in the visual field 21b which is not enlarged. The relationship between the photographing visual field and the width of the sample flow will be described in detailed with reference to Figs. 9A to 9D. Figs. 9A to 9C respectively illustrate some methods of changing the photographing visual field and the way of letting the sample fluid to flow. In each method, the detection region 22, the photographing region 21 and/or the sample flow 23 are switched between the low magnification mode and the high magnification mode. According to the method shown in Fig. 9A, the width of the sample flow 23 is not changed between the low magnification mode and the high magnification mode, but the flow rate and the thickness of the sample are switched. The width of the sample flow 23 is set to be larger than the width of the photographing region 21 in either mode. The width of the detection region 22 is changed to substantially coincide with the width of the photographing region 21. In this method, only a part of the sample flow discharged in the flow cell 1 passes through the photographing region 21. By having investigated the proportion of the sample flow that passes through the photographing region 21, the volume of the sample liquid that is passing through the photographing region can accurately be known. Further, as the width of the detection region 22 is changed to the same as that of the photographing region 21 at the time when switching the mode, the particles detected in the detection region 22 always pass through the photographing region 21. Only the flow rate and the thickness of the sample flow are controlled, and the width of the same is not required to accurately coincide with the photographing region. Since only the central portion of the sample flow 23 is measured, the flow is required only to have a constant speed and a constant thickness, and the structure of the flow cell 1 can be simplified. Further, since the sample flow 23 passes uniformly over the entire width of the photographing region 21 in either mode, the area of the photographing region 21 can effectively be used, and the efficiency of the analysis can be improved. Moreover, as the sample flow 23 is large in width, the flow passage can be enlarged and hardly clogged even when large particles are contained in the sample flow 23. Also in the method shown in Fig. b, the width of the sample flow 23 is not changed between the low magnification mode and the high magnification mode, but the flow rate and the thickness are switched. The width of the sample flow 23 is made to be slightly narrower than that of the photographing region 21b employed in the low magnification mode, through setting of the dimensions of the discharge port 19 and the guides 16 of the nozzle. The width of the detection region 22 is changed to substantially coincide with the width of the photographing region 21. In the this method, only a part of the sample liquid discharged in the flow cell 1 passes through the photographing region 21a in the high magnification mode. By having investigated the proportion of the sample liquid that passes through the photographing region 21a, the volume of the sample that passes through the photographing region can accurately be known. Since the photographing region 21a and the detection region 22a have substantially the same width, the particles detected in the detection region 22a always pass through the photographing region 21a. Further in the low magnification mode, as all the sample liquid discharged in the flow cell 1 passes through the photographing region 21b, the volume of the sample liquid passing through the photographing region can be known more accurately. In the high magnification mode in which the depth of focus of the microscope is shallow, only the central portion of the sample flow 23, that flows at a constant speed and has a uniform thickness, is measured. Therefore, image blur occurring due to unevenness of the thickness can be prevented, and analysis of even particles having very small sizes can precisely be performed. Moreover, since only the flow rate and the thickness of the sample liquid flow are controlled, the necessity of accurately regulating the width can be eliminated, and the structure of the flow cell 1 can be simplified. Further, the sample flow 23 uniformly passes substantially over the entire width of the photographing region 21 in either mode, accordingly, the area of the photographing region 21 can effectively be used, and the analysis can be made efficiently. Also in the method shown in Fig. 9C, the width of the sample flow 23 is not changed between the low magnification mode and the high magnification mode, but the flow rate and the thickness are switched. The width of the sample flow 23 is made slightly narrower than the width of the photographing region 21a employed in the high magnification mode. The width of the detection region 22 is not changed even when the mode is changed. In this method, in either mode, all the sample liquid discharged in the flow cell 1 passes through the photographing region 21, and therefore, the volume of the sample liquid passing through the photographing region can accurately be known. Further, the width of the detection region 22 is not required to be changed, so that the optical system for detection can be simplified, the apparatus can be reduced in size and the adjustment procedure can be simplified. Since only the flow rate and the thickness of the flow of the sample liquid are controlled, the necessity of accurately regulating the width can be eliminated, and the structure of the flow cell 1 can be simplified. Further, as the sample flow 23 uniformly passes through substantially the overall all width of the photographing region 21b in the high magnification mode, the area of the photographing region 21 can effectively be used, and the analysis can efficiently be performed. Since the width of the sample flow 23 is kept narrow also in the low magnification mode, the sample flow 23 can stably flow even at a high speed, and the analysis can efficiently be performed. Moreover, because the particles do not pass through the ends of the photographing region 21b, the overall shapes of the particles can be photographed, and the image analysis can accurately be performed. In the method shown in Fig. 9D, the width, the flow rate and the thickness of a sample flow 123 are switched- between the low magnification mode and the high magnification mode. The width of the sample flow 123 is set to be slightly smaller than that of a photographing region 122 in either mode. The width of a detection region 122 is not changed even when the mode is changed. The width, the thickness and the flow rate of the sample liquid are, in this case, determined as follows. In the high magnification mode, the sample liquid flows with its width slightly narrower than that of a photographing region 121a. The thickness of the sample liquid is made substantially coincide with the thickness which can be photographed with the microscope. In the low magnification mode, the sample liquid flows with its width slightly narrower than that of a photographing region 121b. The thickness of the sample liquid is made substantially coincide with the thickness which can be photographed with the microscope. The measuring method shown in Fig. 9D has the following characteristic features. All the sample liquid discharged in the flow cell passes through the photographing region 121 in either mode, and therefore, the volume of the sample liquid passing through the photographing region can accurately be known. Since the detection region 122 is not required to be changed of its width, the optical system for detection can be simplified, the apparatus can be reduced in size and the adjustment procedure can be simplified. Further, as the sample liquid 123 uniformly flows over the entire width of the photographing region 121, the area of the photographing region 121 can effectively be used, and the analysis can efficiently be performed. Since the particles do not pass through the ends of the photographing region 121, the entire shape of the particles can be photographed, and the image analysis can accurately be performed. Modifications of the flow cell will now be described with reference to Figs. 10 to 14. At the outset, a method of changing the flow of the sample liquid by means of the flow cell 1 of the apparatus according to the first embodiment will be described. According to the foregoing methods shown in Figs. 9A to 9C, the width of the sample flow is not changed regardless of the magnification, but only the flow rate and the thickness are changed. In the flow cell 1 of the first embodiment shown in Fig. 6, the width of the sample liquid is determined depending upon the width of the guides 16 of the nozzle 3, and it is kept constant even if the flow rate of the sample fluid is changed. Therefore, increase in the flow quantity of the sample liquid causes the thickness of the sample flow to be increased while the width being kept constant. That is, in the case of the methods shown in Figs. 9A to 9C, switching between the large magnification mode and the low magnification mode can be made relatively easily by adequately changing the flow quantity of the sheath liquid and that of the sample liquid. In the method shown in Fig. 9D, it is necessary to change the width, the flow rate and the thickness of the sample flow depending upon the magnification. The flow cells shown in Figs. 10 to 14 enable also the width of the sample flow to be varied. The flow cell shown in Fig. 10 is different in structure from the flow cell 1 according to the first embodiment in that double guides and double flow passages, both of which have different lengths, respectively, are provided on a nozzle. That is, on the inside of outer guides 16a, shorter guides 16b are disposed. In the case of a low magnification, a sample flow is formed with its width determined by the distance between the two outer guides 16a. When the magnification is large, another sample flow is formed with its width determined by the distance between the two inner guides 16b. The distances between the respective pairs of the guides are regulated to be adaptable to the width of the sample flow in the high magnification and to that in the low magnification. The widths of a photographing region, a detection region and the sample flow in a measuring portion are set as shown in Fig. 9D. The structure of the nozzle 3a in which the double guides are provided will be described in detail with reference to Fig. 11. The nozzle 3a is composed of a main body 33 which is integrally formed with parallel guides 16b, covers 34 and 35 which are joined to upper and lower sides of the body 33, respectively, and a pair of parallel outer guides 16a which are joined to both lateral sides of the body, respectively. When a magnification is low, the sample liquid is allowed to flow through only a single fluid passage 36 which is formed in the body 33 to open between the two guides 16b, and the sample flow has a width which is equal to the distance between the inner guides 16b. When the magnification is high, the sample liquid is allowed to flow from both of the flow passage 36 and flow passages 37 which are formed in both sides of the body 33. Accordingly, the sample flow has a width which is equal to the distance between the two outer guides 16a. These the flow passages are switched under the control of electromagnetic valves (not shown in the figure) connected to the corresponding flow passages. Fig. 12 shows a modification of the nozzle, in which only one sample passage is provided. The nozzle comprises a main body 38, inner guides 16C which are joined to both sides of the body 38 at an angle thereto, respectively, and guides 16a which are joined to the outsides of the guides 16C, respectively. That is, the nozzle 3b is composed of the pipe-like nozzle main body 38 which is in a hollow, square cross sectional shape, the pair of parallel, plate-like guides 16a which are respectively disposed on the both sides of an outlet port of the nozzle body 38, and the pair of plate-like guides 16C which are respectively disposed inside the parallel, plate-like guides 16a at the angle with their ends directed inwardly. When a magnification is low, the quantity of the sample flow is small, therefore, it flows along the inner guides 16C as indicated by reference numeral 39, and the width of the sample flow 39 is equal to the distance between the ends of the inner guides 16C. When the magnification is high, the quantity of the sample liquid flow is enlarged, therefore, it flows over tapered portions of the inner guides 16C to flow along the outer guides 16a, and the width of a sample flow 40 is equal to the distance between the outer guides 16a. The nozzle 3b shown in Fig. 12 has no necessity of switching the sample fluid passage, and the switching of the sample flow can be performed easily as compared with the nozzle 3a shown in Fig. 11. Also in the case of the nozzle 3b shown in Fig. 12, similarly to the embodiments described above, the sample liquid is discharged from an outlet port in the end of the nozzle body 38. A sheath flow is formed in the flow passage, and the sample liquid flows at a constant speed through the flow passage. At this time, if the quantity of supply of the sample liquid is more than a predetermined level, the sample liquid flows from the outlet port in the end of the nozzle body 38 in such a manner that it moves over the inner guides 16C which are provided to be mutually inclined. The sample liquid, which has moved over the guides 16C, flows along the outer guides 16a as shown in Fig. 13. Therefore, the width of the sample flow 40 in the case depends upon the distance between the guides 16a. Similarly, the quantity of the flow of the sample liquid to be introduced through the sample liquid supply port is reduced during the examination or prior to beginning the examination of a next sample. This operation causes the sample liquid to flow along the inside of the guides 16C without moving over them as shown in Fig. 1 . In this manner, the shape of the sample flow passing through the flow passage can be changed from the sample flow 40 shown in Fig. 13 to the sample flow 39 shown in Fig. 14. That is, the width of the sample flow can be controlled in two sorts by means of the flow quantity of the sample liquid. Further, when the quantity of the flow of the sheath liquid and that of the sample liquid are changed while keeping the ratio of them constant, only the flow rate can be controlled without changing the shape of the sample flow.

Claims: A flow cell apparatus for measuring particles (20) suspended in a liquid, comprising means (10, 27 to 29) for supplying a sample liquid (25) to be measured, means (7, 11) for supplying a sheath liquid (24), flow cell means (2) for defining a flow passage (1) with a transparent measuring portion (1c) providing an unobstructed view of the flow passage (1) in at least one transversal direction thereof, nozzle means (3, 3a, 3b) provided in fluid communication with the sample liquid supplying means (10, 27 to 29) and comprising a nozzle body (17; 33 to 35), and at least one discharge port (19; 36, 37) formed at one end of said nozzle body (17; 33 to 35) and disposed in the flow passage (1) in a spaced relation from an inner wall of the flow passage (1) for causing the sample liquid (25) to flow into the flow passage (1) of said flow cell means (2), and first guide plates (16, 16a) disposed on first opposite sides of said discharge port (19; 36, 37) for guiding the sample liquid (25) from said discharge port (19; 36, 37) whereby the sheath liquid supplying means (7, 11) are so arranged in fluid communication with the flow passage (1) of said flow cell means (2) at a position upstream said discharge port (19; 36, 37) with respect to the flow of the sample liquid (25) as to cause the sheath liquid (24) to surround the sample liquid (25) from the discharge port (19; 36, 37) to form a sheath flow, said nozzle body end is tapered to said discharge port (19; 36, 37) to have on second opposite sides thereof tapered surfaces, whereby said second opposite sides are different from said first opposite sides, and said first guide plates (16, 16a) are tapered to their ends in downstream direction with respect to the flow of the sample liquid (25), characterized in that the tapered surfaces of the nozzle body (17; 33 to 35) terminate between said first guide plates (16, 16a), whereby the first guide plates (16, 16a) are higher than a height of the discharge port (19; 36, 37) at the terminating end of the nozzle body (17; 33 to 35) to stabilize a sample liquid flow (123) from the discharge port (19; 36, 37) at a constant width in at least a direction perpendicular to a direction of the unobstructed view in said measuring portion (1c), and the flow passage (1) has a constant width for preventing change in the width of the sample liquid flow (123). The apparatus according to claim 1, characterized in that said flow cell means (2) further includes means provided upstream said measuring portion (1c) for narrowing the sheath flow in the unobstructed view direction of said measuring portion to increase a velocity of the sample liquid flow (123) in the sheath flow and reduce a thickness of the sample liquid (25) in the unobstructed view direction to form a flat flow. The apparatus according to claim 1, characterized in that said nozzle means (3, 3a, 3b) further includes means for changing the width of the sample liquid flow in accordance with a mesuring range in said measuring portion (1c). The apparatus according to claim 2, characterized in that said sheath flow narrowing means is set to increase the velocity of the sample liquid flow (123) to 1000 mm/second or higher. The apparatus according to claim 2, characterized in that said sheath flow narrowing means is set to reduce the thickness of the sample liquid (25) for photographing particles (20) of the sample liquid through a microscope in said measuring portion (1c). The apparatus according to claim 2, characterized in that said sheath flow narrowing means comprises a portion of said flow passage (1) having a height in the unobstructed view direction, which is gradually decreased downstream. The apparatus according to claim 3, characterized in that said nozzle means (3, 3a, 3b) further includes means for changing the width of the sample liquid flow (123). The apparatus according to claim 3, characterized in that said flow width changing means comprises a plurality of discharge ports (19; 36, 37) of said nozzle means (3, 3a, 3b), which are individually and selectively in communication with said sample liquid supply means (10, 27 to 29). The apparatus according to claim 4, characterized in that said flow width changing means comprises fixed second guide plates (16c) which are respectively provided on both sides of said discharge port (19; 36, 37) in a widthwise direction of said flow passage (1), said fixed second guide plates (16c) being each formed to allow the sample liquid (25) more than a certain flow rate to flow over said fixed second guide plates (16c). The apparatus according to claim 1, characterized in that said flow passage (1) of said flow cell means (2) includes a parallel flow passage portion which passes through said measuring portion (1c) and has a height substantially constant in the unobstructed view direction, and a flow contraction passage portion (1b) which is disposed upstream said parallel flow passage portion and has a height gradually decreased toward said parallel flow passage portion. The apparatus according to claim 10, characterized in that said flow passage (1) of said flow cell means (2) is further provided with a deceleration flow passage portion (1d) which is disposed downstream said parallel flow passage portion and increased in height gradually from said parallel flow passage portion. The apparatus according to claim 11, characterized in that said flow passage (1) of said flow cell means (2) is further provided with another parallel flow passage portion (1a) which has a substantially constant height and is disposed upstream said flow contraction passage portion (1b). The apparatus according to claim 10, characterized in that the height of said parallel flow passage portion is set in accordance with a depth of focus of a microscope to be used in measurement. The apparatus according to claim 10, characterized in that said flow passage (1) of said flow cell means (2) is defined by plate-like members joined together, said flow contraction passage portion (1b) is defined by a pair of flow contracting plates, and said flow contracting plates each have a trapezoidal cross sectional shape in a direction of the sample liquid flow (123) and are secured in said flow passage (1) to face each other. The apparatus according to claim 14, characterized in that said plate-like members are transparent. The apparatus according to claim 10, characterized in that said nozzle means (3, 3a, 3b) has three discharge ports (19; 36, 37) arranged in a widthwise direction of said flow passage (1), two discharge ports at both sides of said three discharge ports are selectively in communication with said sample liquid supply means (10, 27 to 29) to change the width of the sample liquid flow (123). The apparatus according to claim 16, characterized in that the central one of said three discharge ports is provided at both sides thereof in the widthwise direction of said flow passage (1) with a pair of third elongated guide plates (16b) which each extend substantially in parallel to the sample liquid flow (123), and said first guide plates (16a) are larger in length in the direction (25) of the sample liquid flow (123) than said third guide plates (16b). The apparatus according to claim 10, characterized in that said nozzle means (3, 3a, 3b) further includes a second pair of elongated guide plates (16c) respectively provided on both sides of said discharge port (19; 36, 37) in the widthwise direction of said flow passage (1), said second guide plates (16c) are smaller in length in the direction (25) of the sample liquid flow (123) than said first guide plates (16a) and inclined inwardly toward each other to form the sample liquid flow of a small width, and said first guide plates (16a) are disposed outside the respective second guide plates (16c) and extend substantially in parallel to the direction (25) of the sample liquid flow (123) to form the sample liquid flowing over said second guide plates (16c) into a flow of a constant width. The apparatus according to claim 18, characterized in that each of said second guide plates (16c) is tapered in a downstream direction.

Inventors: HORIUCHI HIDEYUKI, ISHII MASAHARU, MATSUMOTO MASAETSU, MIYAKE RYO, OHKI HIROSHI, SAKURABA SHINICHI, YABE RYOHEI, YAMAZAKI ISAO, HORIUCHI, HIDEYUKI, ISHII, MASAHARU, MATSUMOTO, MASAETSU, MIYAKE, RYO, OHKI, HIROSHI, SAKURABA, SHINICHI, YABE, RYOHEI, YAMAZAKI, ISAO
Applicants: HITACHI LTD, HITACHI, LTD.
Application year: 1994
Main classification: G01N21
IPC codes: G01N21, G01N15, G01N33